Ribosome Stalling of N-Linked Glycoproteins in Cell-Free Extracts

Ribosome display is a powerful in vitro method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation. The ability to generate glycosylated ribosome–nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free N-glycoprotein synthesis. This integration enabled a first-in-kind method for ribosome stalling of target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the observation that encoding mRNAs remained stably attached to ribosomes provides evidence of a genotype–glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-glycoproteins with advantageous biological and biophysical properties.


Supplementary Methods
Plasmids. All plasmids constructed in this study were made using standard cloning protocols. For in vivo expression, plasmid pET-Im7 N58 -SecM17 was constructed by inserting the PCR-amplified product encoding Im7 N58 in place of the gene encoding scFv13-R4 in plasmid pET-scFv13-R4-SecM17 1 . Plasmid pET-DQNAT scFv13-R4-SecM17 was constructed by similar replacement but with a PCR-amplified product encoding DQNAT scFv13-R4 in which the N-terminal DQNAT motif was introduced via the forward primer. Plasmid pET-Im7 N58 was constructed by PCR amplification of the gene encoding Im7 N58 from plasmid pTrc99S-YebF-Im7 N58 2 and subsequent Gibson isothermal assembly 3 of the resulting PCR product into the amplified plasmid backbone of pET28a. For in vitro expression, plasmid pJL1-Im7 N58 -SecM17 was constructed by PCR amplification of the gene encoding Im7 N58 -SecM17 from plasmid pET-Im7 N58 -SecM17 and Gibson assembly of the resulting PCR product into the amplified pJL1 plasmid backbone. Plasmids pJL1-scFv-HER2 DQNAT -SecM17 and pJL1-DQNAT PD-SecM17 were constructed by PCR amplification of the genes encoding scFv-HER2 DQNAT from plasmid pTrc99S-YebF-scFv-HER2 DQNAT 2 and PD from plasmid pJL1-PD 4xDQNAT 4 , respectively, and inserting the resulting PCR products in place of Im7 N58 in amplified plasmid pJL1-Im7 N58 -SecM17 via Gibson assembly. The same PCR products were used to construct plasmids pJL1-scFv-HER2 DQNAT and pJL1-DQNAT PD by Gibson assembly into plasmid pJL1. Additional plasmids constructed previously and used in this study included: plasmid pSN18 for expression and purification of CjPglB 5 ; plasmid pSF-CjPglB for selective enrichment of CjPglB in cell-free extracts 6 ; plasmid pMW07-pglΔB for expression and extraction of CjLLOs 7 ; and plasmid pET28-ColE7 H569A for expression and purification of ColE7 2 . All plasmids were confirmed by DNA sequencing at the Biotechnology Resource Center (BRC) Genomics Facility (RRID:SCR_021727) in the Cornell Institute of Biotechnology.
Ribosome isolation. Cells transformed with the pET28a-derived constructs were grown in 100-mL cultures and induced with 1 mM IPTG at an Abs600 ∼0.5 and grown at 30 °C for an additional 30 min. Following expression, two buffer C (20 mMTris-HCl (pH 7.5), 50 mM NH4Cl, 25 mM MgCl2) ice cubes were added to each culture flask, rapidly shaken for 1 min on ice and incubated on ice for an additional 30 min. Next, cells were pelleted by centrifugation for 30 min at 4 °C and 10,000×g and resuspended in 600 μL of cold buffer C. Cells were lysed by three cycles of freeze-thawing in liquid nitrogen followed by the addition of three 30-μL aliquots of lysozyme (Novagen), where the stock lysozyme solution was diluted 50-fold in cold buffer C and each lysozyme addition was followed by a 20 min incubation at 4 °C, and finally three additional freeze-thawing cycles. Ribosomes were isolated from these soluble fractions according to previously published procedures  Figs. 1 and 2).
For visualizing the separated protein samples, gels were stained with Coomassie G-250 stain (Bio-Rad) following the manufacturer's protocol. For Western blot analysis, the separated protein samples were then transferred to nitrocellulose membranes using a semi-dry apparatus. Following transfer, the membranes were blocked with 5% milk (w/v) in TBST (1x TBS, 0.1% Tween 20) and subsequently probed for 1 h with one of the following: horseradish peroxidase (HRP)-conjugated anti-DDDDK antibody (Abcam, cat # ab49763) that recognized the FLAG epitope tag; the C. jejuni heptasaccharide glycanspecific antiserum hR6 (kindly provided by Markus Aebi); or the mouse mAb FB11 (ThermoFisher; cat # MA1-7388) that specifically recognizes F. tularensis LPS. Goat antirabbit IgG (HRP) (Abcam, cat # ab205718) was used as the secondary antibody to detect hR6 antiserum while HRP-conjugated anti-mouse IgG (Abcam, cat # ab97023) was used as the secondary antibody to detect FB11. After washing five to six times with TBST for 5 min, the membranes were visualized using a ChemiDoc MP Imaging System (Bio-Rad).

ELISA.
Binding activity for ribosome-tethered Im7 N58 and scFv-HER2 DQNAT was determined by standard ELISA. Briefly, treated Costar 96-well ELISA plates (Corning) were coated overnight at 4 °C with 50 μL of 5-μg/mL in-house prepared ColE7 in PBS for ribosome-tethered Im7 N58 or the same amount of commercial extracellular domain Plates were washed three times and then developed using 50 μL 1-Step Ultra TMB-ELISA substrate solution (ThermoFisher). A similar ELISA protocol was followed for detecting RNC complexes displaying glycosylated DQNAT PD except that the plates were coated with 50 μL of 5-μg/mL mouse mAb FB11 in PBS and were subsequently detected with HRPconjugated anti-mouse IgG.
Statistical analysis and reproducibility. To ensure robust reproducibility of all results, experiments were performed with multiple biological replicates and technical measurements. Sample sizes were not predetermined based on statistical methods but were chosen according to the standards of the field (at least three independent biological replicates for each condition), which gave sufficient statistics for the effect sizes of interest. All data were reported as average values with error bars representing standard deviation. Statistical significance was determined by unpaired t test with Welch's correction (*p < 0.05, **p < 0.01; ns, not significant). All graphs were generated using